Transplantation of mobilized hematopoietic stem cells (HSCs) has become a standard of care for hematologic malignancies and blood diseases such as leukemia, bone marrow failure syndromes, and multiple myeloma. Novel methods promoting HSC engraftment are needed to improve the efficacy and safety of stem cell and gene therapies, especially for those patients where conventional HSC mobilization regimens are not effective or myeloablative conditioning leads to morbidity and mortality. To this end, developing more effective approaches for HSC mobilization and allowing increased engraftment and stable chimerism of quantity-limited CD34+ human HSCs or gene therapy-corrected HSCs could significantly impact on future stem cell transplantation practice for hematologic malignancies. The Rho family GTPase Cdc42 plays critical roles in regulating cytoskeleton dynamics, 1 integrin-mediated adhesion, and SDF-1a induced directional migration, functions that are essential for HSC residence in the bone marrow niche. In preliminary studies we have characterized a conditional gene targeted mouse model and identified a Cdc42 specific small molecule inhibitor to define the role of Cdc42 in HSC residency in the bone marrow. We show that gene targeting or pharmacological targeting of Cdc42 causes massive mobilization of functional HSCs, transient opening of bone marrow niche, and effective engraftment of syngeneic or autolagous transplanted HSCs. Our results suggest that Cdc42 constitutes a critical nodal of intracellular signal flows involved in HSC maintenance in the bone marrow, and lead to our central hypothesis that Cdc42 is essential for HSC residence in the BM niche and represents a useful target for HSC engraftment, properties useful for improving bone marrow transplant efficacy. The aims of the proposed studies are (1) to define the mechanism of action of lead Cdc42 inhibitor and improve the lead efficacy by medicinal chemistry, and (2) to establish a proof of principle of Cdc42 targeting as a non- myeloablative conditioning regimen for HSC engraftment in mouse models. By achieving the goals of these early stage drug discovery studies, we may establish a new method for blood stem cell transplantation that may significantly impact on future cell therapies for blood malignancies.